Hi, in our lab we have been using S-35 labelled oligo probes and radioactive ISH for years, but would like to switch to DIG labelled oligos. We have tried numerous times with different protocols, but nothing seems to work. Any suggestions? We are using ssDNA 45-mer oligos. Does anyone have any working protocols you wouldn't mind sharing?
I also used isotope probes for many years but then switched to DIG-labeled probes. This paper (free access) describes both and cites relevant methods papers.
The main thing I notice is that your oligos are very short. In my experience, we got good results with probes much longer (>300 to 1000 bp). Longer probes are more specific, and also carry more DIG. So this may help both with quality and quantity of signal. Good luck with your experiment.
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